New animal model of chronic gout reproduces pathological features of the disease in humans

Objectives Gout, as the most prevalent form of inflammatory arthritis, necessitates the use of animal models to investigate the molecular mechanisms involved in its development. Therefore, our objective was to develop a novel chronic mouse model of gout that more closely mimics the progression of gout in humans. Methods A novel chronic mouse model of gout was established by a simple method, which does not require high technical proficiency, predominantly involves daily intraperitoneal injections of potassium oxonate for approximately 4 months, combined with a high fat-diet and injections of acetic acid into the hind paws to facilitate the formation of monosodium urate (MSU). Arthritis scores and paw oedema were assessed, behavioural tests were conducted, and histopathological and imaging evaluations of the arthritic paw joints were performed. Results After 4 months of induction, mice in the model group exhibited noticeable increases in arthritis severity, joint and cartilage damage, as well as bone erosion. Gomori’s methenamine silver stain revealed the presence of MSU crystal deposition or tophi in the paw joints or ankle joints of up to 37.9% of the model mice (11 out of 29 mice). Moreover, treatment with benzbromarone effectively prevented the further development of gout or tophi formation in model mice. Conclusions Our model more accurately replicates the pathological features of gouty arthritis compared with gout induced by MSU crystal injections. Therefore, it is particularly suitable for further investigations into the pathogenesis of gout and also serves as a valuable platform for screening potential antigout agents.

Supplemental material: Immunohistochemical analysis, cytokine enzyme linked immunosorbent assay (ELISA), quantitative real-time PCR, western blot analysis and in vivo lucigenin bioluminescence imaging of NADPH oxidase activity.

Cytokine enzyme linked immunosorbent assay (ELISA)
The ankle joints and paws of mice were dissected, weighed and placed into cold phosphate buffered saline (PBS) containing protease inhibitor cocktail (Roche Diagnostics) at a final concentration as recommended by the manufacturer, then homogenized with a QM100 bead miller (Wuzhou Ding Chong, Beijing, China) for 2 min at room temperature.The homogenates were centrifuged twice at 15,000 r.p.m for 20 min at 4 ℃ to remove tissue and cell debris and the resulting supernatants were aliquoted and stored at -80 ℃.The pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and anti-inflammatory cytokine (IL-10) in the supernatants were measured using Abcam's ELISA kit (Abcam, Cambridge, UK), according to the manufacturer's instructions.Briefly, diluted samples or standards were added to 96-well plated precoated with affinity purified antibody specific for mouse TNF-α, IL-1β, IL-6 and IL-10, respectively.The wells were washed and biotinylated anti-mouse TNF-α, IL-1β, IL-6 and IL-10 antibodies were added as appropriate.After washing away unbound biotinylated antibodies, horseradish peroxidase (HRP)-conjugated streptavidin was BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) RMD Open doi: 10.1136/rmdopen-2023-003499 :e003499.9 2023; RMD Open , et al.Wang J pipetted to the wells and tetramethylbenzidine (TMB) was used as a substrate.The intensities of the color detected at 450 nm reflected the level of cytokines present.

Quantitative real-time PCR
Total RNA was extracted from hind paws and ankle joints using RNApre pure Tissue Purification kit (Tiangen, China) following the manufacturer's instructions.First-strand cDNA was synthesized by reverse transcription of the total RNA using the transcript II RT kit (Taingen, China).The cDNA products and corresponding primers were used for iQ SYBR Green Supermix-quantitative real-time PCR (Bio-rad, California, USA) to assay the levels of TNF-α, IL-1β, IL-6 and IL-10.The primers were designed based on the mRNA sequences in GenBank and synthesized by Shanghai Shenggong Botechnology (Shanghai, China).Quantitative RT-PCR was carried out on a Lightcycler96 detection system (Roche, Basel, Switzerland) with mouse β-actin as an internal control.The fold change in the gene expression levels of target genes were calculated with values for normalization to GAPDHusing the 2 -ΔΔCt comparative cycle threshold method.Amplification was performed in the following cycling conditions: 94 ℃ for 15 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 30 s with a total of 40 cycles.Primer sequences were summarized in online supplemental Table S1.

Western blot analysis
Protein samples were prepared in the same way as for ELISA analysis, and 30 μg protein was loaded on each lane of a 10% SDS-PAGE gel.Following separation, BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) RMD Open doi: 10.1136/rmdopen-2023-003499 :e003499.9 2023; RMD Open , et al.Wang J

BMJ
Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) staining, the expression of different inflammatory factors in the synovial tissue of paw and ankle joints was scored semi-quantitatively on a four-point scale independently and blindly by two individual pathologists, and the average of their scores was calculated.A score of 0 represented minimal expression; 1, mild expression; and 2, moderate expression; whereas 3 represented abundant expression of inflammatory factors.